The present invention relates to a photocleavable mass tag and the use thereof, and more particularly to a photocleavable mass tag, which may be easily photocleaved by a predetermined laser wavelength to thus release a specific mass-tagged cation so as to be efficiently used for MALDI-TOF (Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight) mass spectrometry and matrix-less LDI-TOF (Laser Desorption/Ionization Time-Of-Flight) mass spectrometry. Also, the present invention relates to a technique for increasing the sensitivity of mass spectrometry using a compound for efficiently forming ions through light irradiation as a mass tag for mass spectrometry.
MALDI-TOF mass spectrometry is a method of analyzing the mass of a sample based on a difference in time of flight depending on m/z of ions generated in a manner in which the sample is crystallized through the addition of a matrix for absorbing UV light and is then ionized through laser irradiation. Such mass spectrometry is useful in the analysis of biopolymers such as proteins, as well as synthetic polymers, additives, and the like, because the absolute mass of a polymer may be measured with high sensitivity in a short time. However, since laser energy irradiated for an ionization process is transferred to the sample via the crystallized matrix to thereby ionize the sample, appropriate selection of a matrix has a great influence on test results. Accordingly, in order to obtain more accurate test results by removing the matrix, matrix-less LDI-TOF mass spectrometry has been devised.
Ferrocene-based novel photocleavable mass tags for forming stable cations have been synthesized. In this regard, Korean Patent Application No. 10-2014-0050964 (entitled: Photocleavable Mass Tag and Use Thereof) discloses the use of ferrocene in which a carbocation at the alpha position of ferrocene is stable, and the structure thereof is shown in FIG. 1. This patent document is incorporated by reference herein because ionization may be efficiently performed through laser irradiation in the absence of a matrix through introduction of a mass-changing group and a reactive group. The mass spectrometry spectrum results of the equimolar mixture of various ferrocene photocleavable mass tags are shown in FIG. 2.
Although the ferrocene-based mass tags are advantageous because of easy synthesis and high photocleavage efficiency compared to trityl derivatives, the solubility thereof in water is low, as in trityl derivatives, resulting in very low conjugation yields upon conjugation thereof with biomaterials such as peptides, nucleic acids, glycans, and the like, having high hydrophilicity and water solubility. Furthermore, with the goal of increasing the yield of conjugation with a biomaterial, even when a mixture of an organic solvent and water is used, the proportion of the organic solvent has to be increased in order to dissolve the hydrophobic ferrocene or trityl tag. During the conjugation, the three-dimensional structure of a biomaterial such as a protein or antibody may be deformed, thus losing the specificity of the protein or antibody, which is undesirable. Hence, in order to solve such problems, there is a need for the development of mass tags having high solubility in water.
As conventionally known mass tags, various mass tags, formed by introducing substituents having different masses to triphenylmethyl or trityl groups for relatively efficient formation of cations under specific conditions, and combinatorial synthesis methods thereof are known (Shchepinov et al, Nucl. Acids Symp. Ser. 1999, 42, 107-108). Such methods are characterized in that, under mass spectrometry conditions in which the molecular weight of a predetermined molecule is measured using a difference in time of flight (TOF) after ionization through laser irradiation, an analytical sample may be ionized even without a matrix for aiding the ionization.
There are known documents pertaining to a method of calibrating a mass spectrometer in a similar manner using a trityl derivative (M. S. Shchepinov et al, U.S. Pat. No. 6,734,025 B2, May 2004), an analysis technique using an oligomer library tagged with the above tag (E. M. Southern et al, U.S. Pat. No. 6,780,981 B1, August 2004), a technique for increasing the mass spectrometry sensitivity of a biomaterial using the above tag (M. S. Shchepinov et al, US 2008/0248584 A1), a technique for imaging a biosample using a mass spectrometer and an antibody tagged with the above tag (I. G. Gut, US 2011/0223613), and the like. These documents using MALDI-TOF are incorporated by reference herein.
Accordingly, the present invention is intended to provide a compound, which enables the formation of stable carbocation after photocleavage, has high absorbance at a predetermined laser wavelength (i.e. 355 nm), has a molecular weight of 500 Dalton or less to thus exhibit high solubility in water, has high hydrophilicity due to the presence of a heteroatom in a molecular structure thereof, may be easily synthesized at high yield, and has a structure to which a plurality of mass-changing groups may be easily introduced.
According to the present invention, a 2-alkylsulfanyl-2H-thiochromene derivative may be utilized as a photocleavable mass tag able to release a cation having a specific mass by being easily photocleaved under conditions of MALDI-TOF MS (Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry) in which a laser at 355 nm is applied. Furthermore, the thiochromene derivative according to the present invention is capable of generating cations using only a laser even under matrix-less LDI-TOF MS conditions, unlike conventional MALDI-TOF MS. Accordingly, noise caused by the addition of a matrix may be completely removed, and a polymer such as an antibody may be detected with high sensitivity.
Therefore, the present invention provides a thiochromene-type compound, particularly a 2-alkylthio-2H-thiochromene derivative compound, which is useful as a photocleavable mass tag. The principle thereof is shown in FIG. 3. Specifically, the compound according to the present invention is composed of a UV-absorbing group for absorbing UV light, a reactive group able to react with a biomaterial, a linker that enables the UV-absorbing group and the reactive group to be connected or cleaved through light irradiation, and a mass-changing group that may be substituted through a change in mass.
An aspect of the present invention provides a compound represented by Formula I below:

In Formula I, R1 is a linker having an active reactive group able to easily react with a functional group present on a solid or a biomaterial, such as an amine, thiol, or the like, examples of the active reactive group including a N-hydroxysuccinimide ester group, a N-hydroxysulfosuccinimide ester group, a pentafluorophenyl ester group, a 4-sulfo-2,3,5,6-tetrafluorophenyl ester group, a nitrophenyl ester group, a 2,4,5-trichlorophenyl ester group, a phthalimido ester group, a N-hydroxy-5-norbornen-endo-2,3-dicarboimide ester group, and a maleimide group. The connection portion of the linker may include, for example, an alkyl group, but is not limited thereto, and may be ether-containing alkyl, aryl or heteroaryl. Preferably, the connection portion of the linker is a C1-12 alkyl group, a C6-60 aryl group, or a C2-60 heteroaryl group having at least one heteroatom selected from among N, S and O.
As used herein, the term “alkyl group” refers to a C1-60 linear or branched alkyl group such as linear or branched alkyl, including a methyl group, an ethyl group, a propyl group, an isopropyl group, a N-butyl group, an isobutyl group, a tert-butyl group, a N-pentyl group, or a N-hexyl group, and preferably a C1-12 linear or branched alkyl group.
As used herein, the term “aryl group” refers to a C6-60 aryl group having a hydrocarbon ring, for example, a phenyl group, a naphthyl group, etc., and the term “heteroaryl group” refers to a C2-60 aromatic heteroaryl group containing at least one heteroatom selected from the group consisting of N, S and O at any possible position.
As used herein, the term “active reactive group” refers to a N-hydroxysuccinimide ester group, a N-hydroxysulfosuccinimide ester group, a pentafluorophenyl ester group, a 4-sulfo-2,3,5,6-tetrafluorophenyl ester group, a nitrophenyl ester group, a 2,4,5-trichlorophenyl ester group, a phthalimido ester group, a N-hydroxy-5-norbornen-endo-2,3-dicarboimide ester group, or a maleimide group. Preferably, the active reactive group is a N-hydroxysuccinimide ester group (NHS), a pentafluorophenyl ester group, a nitrophenyl ester group, or a maleimide group, and is more preferably a N-hydroxysuccinimide ester group.
In Formula I, R2 and R3 may be the same as each other, and are independently hydrogen, alkyl, aryl, alkoxy, alkylamino, alkylthio or a fused ring. Here, “alkyl” or “aryl” is as defined above.
As used herein, the term “alkoxy” refers to a linear or branched C1-12 alkoxy group, such as a methoxy group, an ethoxy group, a N-propoxy group, a N-butoxy group, an isobutoxy group, a tert-butoxy group, or a N-pentoxy group.
As used herein, the term “fused ring” refers to a ring obtained via the condensation of a phenyl group or an aromatic heterocyclic group containing at least one heteroatom selected from among O, S and N, at any possible position, with a benzene ring or an aromatic heterocyclic group containing 1 to 3 heteroatoms selected from among oxygen, sulfur and nitrogen atoms, and is any one selected from among, for example, pyrrole, thiophene, indole, furan, imidazole, triazole, diazole, and pyrimidine. Preferably, the fused ring is thiophene, pyrrole, indole, or furan.
In Formula I, Ar is a C6-60 aromatic ring, a heteroaromatic ring, or an extension ring achieved through a combination of heteroaromatic rings. The “heteroaromatic ring” may be a monocyclic or polycyclic hetero ring containing 0, N or S as a heteroatom, the number of carbons of which is not particularly limited, but is preferably 2 to 60. Examples of the heterocyclic group may include, but are not limited to, a thiophene group, a furan group, a pyrrole group, an imidazole group, a thiazole group, an oxazole group, an oxadiazole group, a triazole group, a pyridyl group, a bipyridyl group, a triazine group, an acridyl group, a pyridazine group, a quinolinyl group, an isoquinoline group, an indole group, a benzoxazole group, a benzimidazole group, a benzothiazole group, a benzocarbazole group, a benzothiophene group, a dibenzothiophene group, a benzofuranyl group, a dibenzofuranyl group, and the like. Preferably, the heteroaromatic ring of Ar is benzene, pyrrole, thiophene, indole, furan, imidazole, triazole, diazole, or pyrimidine.
As used herein, the term “extension ring” refers to a fused ring compound of heteroaromatic rings.
When a hetero ring is introduced, the solubility of the tag in water may be increased, and the stability of the thiochromenylium cation that is produced may also be increased.
Another aspect of the present invention provides compounds of Formulas II to V below:

In Formulas II, III, IV and V, R1, R2 and R3 are as defined in Formula I.
In Formulas III and V, R4 and A are each a linker having an active reactive group able to easily react with any reactive group (amine, thiol, etc.) present on a solid or a biomaterial, preferable examples of the active reactive group including a N-hydroxysuccinimide ester group, a N-hydroxysulfosuccinimide ester group, a pentafluorophenyl ester group, a 4-sulfo-2,3,5,6-tetrafluorophenyl ester group, a nitrophenyl ester group, a 2,4,5-trichlorophenyl ester group, a phthalimido ester group, a N-hydroxy-5-norbornen-endo-2,3-dicarboimide ester group, and a maleimide group, and the connection portion of the linker is preferably a C1-12 alkyl group, a C6-60 aryl group, or a C2-60 heteroaryl group containing at least one heteroatom selected from among N, S and O.
In Formula III, n is an integer of 1-12.
Still another aspect of the present invention provides a photocleavable mass tag, and preferably a 2-alkylthio-2H-thiochromene derivative useful for MALDI-TOF or matrix-less MALDI-TOF. Such a derivative is represented by Formulas I to V.
The principle pertaining to the photocleavable mass tag according to the present invention is shown in FIG. 4. When a laser is applied at 355 nm, the alkylthio group at position 2 is subjected to heterolytic cleavage to thus easily form a thiochromenylium cation, which is then easily detected in a mass spectrometer. Thus, ionization may become easy through laser irradiation alone, thereby achieving high precision for MALDI-TOF mass spectrometry and matrix-less LDI-TOF mass spectrometry.
Preferable examples of the 2-alkylthio-2H-thiochromene-type mass tag according to the present invention are compounds 1, 2, and 3 (3a-3d) below.

Compounds 1, 2 and 3 have a fundamental backbone of 2-alkylthio-2H-thiochromene and a NHS group (N-hydroxysuccinimidyl ester) able to react with an amine group at the terminal thereof.
For compound 2, an alkoxy group (MeO—) is introduced to benzene rings substituted at positions 3 and 4 of thiochromene. For compound 3, any functional group R may be introduced to N of the indole ring. The functional group R may be modified with Me, Et, n-Pr, n-Bu, or the like, or may be variously modified in a manner similar thereto.
Yet another aspect of the present invention provides a method of synthesizing a photocleavable mass tag. For example, Synthesis Method 1 is exemplarily illustrated below. The synthesis of the mass tag according to the present invention is not limited to the following method.

In accordance with Synthesis Method 1, thiochromeno[4,3-b]indole-based photocleavable mass tag derivatives (3a-3d) may be obtained with high efficiency. Also, in accordance with Synthesis Method 1, various thiochromene-type compounds may be synthesized at a high yield of about 15% using easily commercially available starting materials.
Preferably, the photocleavable mass tag according to the present invention is indole-introduced 2-alkylthio-2H-thiochromene of Formula 3a, 3b, 3c or 3d below.

Still yet another aspect of the present invention provides a conjugate of a biomaterial and a mass tag represented by Formula Ia below.

In Formula Ia, A is a linker having an active reactive group able to easily react with any reactive group (amine, thiol, etc.) present on a solid or a biomaterial, examples of the active reactive group preferably including a N-hydroxysuccinimide ester group, a N-hydroxysulfosuccinimide ester group, a pentafluorophenyl ester group, a 4-sulfo-2,3,5,6-tetrafluorophenyl ester group, a nitrophenyl ester group, a 2,4,5-trichlorophenyl ester group, a phthalimido ester group, a N-hydroxy-5-norbornen-endo-2,3-dicarboimide ester group, and a maleimide group, and the connection portion of the linker is preferably a C1-12 alkyl group, a C6-60 aryl group, or a C2-60 heteroaryl group containing at least one heteroatom selected from among N, S and O.
Although not limited to the following, R2 and R3 are preferably hydrogen, a C1-12 alkyl, a C6-60 aryl, a C1-12 alkoxy, a C1-12 alkylamino, a C1-12 alkylthio, or a fused ring, and Ar may be an aromatic ring such as benzene, or a heteroaromatic ring selected from among pyrrole, thiophene, indole, imidazole, triazole, diazole and pyrimidine.
The biomaterial may be an antigen, an antibody, a biomarker, a peptide, a nucleic acid, a glycan, a cell tissue, etc., and may include a variety of polymer compounds regardless of the kinds thereof. For example, the molecular weight of a polymer compound is not limited, but may be 200,000 Da or more.
According to the present invention, a thiochromene-type compound can be easily synthesized and has high solubility in water. Also, the thiochromene-type compound according to the present invention, having peculiar photocleavability, can be utilized as a high-sensitivity mass tag not only for MALDI-TOF but also for matrix-less LDI-TOF. Particularly in matrix-less LDI-TOF, mass spectrometry results for various polymers can be obtained with high sensitivity due to the absence of a matrix.